Development of a Solid-Phase Dispersive Extraction Method for Molecularly Imprinted Polymers and LC-MS/MS for Analysis of Clenbuterol Residues in Swine Livers and Kidneys.

BACKGROUND: Clenbuterol (CLB) is approved as a veterinary drug because of its tracheal smooth muscle and uterine relaxant effects. However, if improperly administered for the purpose of fattening livestock, CLB can remain in the organs, which may pose a health hazard to humans. OBJECTIVE: We aimed to examine the combination of molecularly imprinted polymer (MIP) and solid-phase dispersive extraction (SPDE) as a pretreatment method for swine liver and kidney, which contain more coexisting impurities than muscle tissue, and attempted to construct an analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: Swine livers and kidneys were homogenized and extracted using liquid-liquid partitioning with an ethyl acetate-n-hexane (1 + 1) mixture, followed by SPDE using an MIP gel, and measured using LC-MS/MS. For LC-MS/MS, either an absolute calibration method or isotope dilution mass spectrometry (IDMS) was used. For method validation, a recovery test (additive concentrations: 0.05 and 0.5 ng/g) was conducted, and the data were analyzed using one-way analysis of variance (ANOVA). RESULTS: The recoveries (trueness), repeatability, and intermediate precision obtained using absolute calibration were similar to those obtained using IDMS. CONCLUSION: Using MIP-SPDE as a pretreatment method for CLB in swine liver and kidney samples yielded comparable results for absolute calibration and IDMS in LC-MS/MS analysis. HIGHLIGHTS: MIP-SPDE can be used as a pretreatment method to analyze CLB in swine organs with high accuracy.

A LC-MS/MS method for determination of clenbuterol enantiomers in animal tissues and its application to the enantioselective distribution in Bama mini-pigs.

OBJECTIVES: To establish and validate a simple and reliable analytical method for separation and determination of clenbuterol enantiomers (R-(-)-clenbuterol & S-(+)-clenbuterol) in animal tissues, and apply it to the enantioselective distribution of clenbuterol in Bama mini-pigs. METHODS: A LC-MS/MS analytical method was developed and validated in positive multiple reaction monitoring mode with electrospray ionization. After perchloric acid deproteinization, samples were pretreated only by one step liquid-liquid extraction using tert-butyl methyl ether under strong alkaline condition. Teicoplanin was used as chiral selector and 10 mM ammonium formate methanol solution was used as mobile phase. The optimized chromatographic separation conditions were completed in 8 min. Two chiral isomers in 11 edible tissues from Bama mini-pigs were investigated. RESULTS: R-(-)-clenbuterol and S-(+)-clenbuterol can be baseline separated and accurately analyzed with a linear range of 5-500 ng/g. Accuracies ranged from -11.9-13.0% for R-(-)-clenbuterol and -10.2-13.2% for S-(+)-clenbuterol, intra-day and inter-day precisions were between 0.7 and 6.1% for R-(-)-clenbuterol and 1.6-5.9% for S-(+)-clenbuterol. R/S ratios in edible tissues of pigs were all significantly lower than 1. CONCLUSIONS: The analytical method has good specificity and robustness in determination of R-(-)-clenbuterol and S-(+)-clenbuterol in animal tissues, and can be used as a routine analysis method for food safety and doping control. There is a significant difference in R/S ratio between pig feeding tissues and pharmaceutical preparations (racemate with R/S ratio of 1), which makes it possible to identify the source of clenbuterol in doping control and investigation.

Nanosensors: A smart remedy for early detection of clenbuterol contamination in food.

Veterinary drugs which are primarily meant for livestock treatment have now been categorised under potential food contaminant due to its unregulated usage and abuse. Their over usage by animal workers lead to production of contaminated animal-based food products which contain veterinary drug residues. These drugs are also misused as growth promoters to enhance the muscle to fat ratio in human body. This review highlights the misuse of such a veterinary drug; Clenbuterol. In this review, we have comprehensively discussed the usage of nanosensors to detect clenbuterol in food samples. Colorimetric, fluorescent, electrochemical, SERS and electrochemiluminescence are major categories of nanosensors that have been utilized for this purpose. The mechanism through which these nanosensors detect clenbuterol have been discussed in detail. The limit of detection and recovery percentage values of each nanosensor have been compared. This review will impart significant information on various nanosensors for clenbuterol detection in real samples.

Determination of ß2-Agonist Residues in Fermented Ham Using UHPLC-MS/MS after Enzymatic Digestion and Sulfonic Resin Solid Phase Purification.

ß2-agonists are a class of synthetic sympathomimetic drugs with acute poisoning effects if consumed as residues in foods. To improve the efficiency of sample preparation and to overcome matrix-dependent signal suppression in the quantitative analysis of four ß2-agonists (clenbuterol, ractopamine, salbutamol, and terbutaline) residues in fermented ham, an enzyme digestion coupled cation exchange purification method for sample preparation was established using ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS). Enzymatic digests were subject to cleanup treatment on three different solid phase extraction (SPE) columns and a polymer-based strong cation resin (SCR) cartridge containing sulfonic resin was found to be optimal compared with silica-based sulfonic acid and polymer sulfonic acid resins based SPEs. The analytes were investigated over the linear range of 0.5 to 10.0 µg/kg with recovery rates of 76.0-102.0%, and a relative standard deviation of 1.8-13.3% (n = 6). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 µg/kg and 0.3 µg/kg, respectively. This newly developed method was applied to the detection of ß2-agonist residues in 50 commercial ham products and only one sample was found to contain ß2-agonist residues (clenbuterol at 15.2 µg/kg).

Production of a matrix certified reference material for measurement and risk monitoring of clenbuterol in mutton.

A novel matrix certified reference material (CRM) for clenbuterol in mutton (GBW 10216) was developed to assist measurement and risk monitoring of clenbuterol in mutton. The candidate CRM raw samples were obtained by oral administration of clenbuterol and investigating the pharmacokinetics of clenbuterol in sheep. A high-precision isotope dilution coupled with liquid chromatography tandem mass spectrometry (LC-ID-MS/MS) method was established and assigned the value of clenbuterol in mutton powder through combined detection of nine inter-laboratories. The certified value with expanded uncertainty was 21.1 ± 2.2 µg/kg (k = 2, 95% confidence) for clenbuterol in mutton. The prepared matrix CRM was sufficiently homogeneous between and within bottles. The long-term stability of clenbuterol in mutton powder was evaluated for 12 months at -20℃ and short-term stability for 7 days at 4℃ and 50℃. The uncertainties originating from characterization, homogeneity, and stability were systematically analyzed and evaluated. The prepared matrix CRM can be applied for proficiency testing and nationwide risk monitoring programs to guarantee the accuracy and comparability of clenbuterol measurement results in mutton.

Quantification and enantiomeric distribution of clenbuterol in several bovine tissues using UHPLC-tandem mass spectrometry: Evaluation of a risk factor associated with meat contamination.

Clenbuterol (Clb) (4-amino-α-[(tert-butylamine) methyl]-3,5-dichlorobenzyl alcohol) is a sympathomimetic agent that exhibits ß2-agonist activity. It is applied as a bronchodilatory, tocolytic, and mucolytic agent and is authorized for clinical management in both human and veterinary therapeutics as a racemic mixture. However, its use is strictly prohibited in animals destined for food production in countries in the European Union and in the United States and Mexico, among many others. The R-(-) enantiomer in clenbuterol stimulates ß2-receptors, whereas the S-(+) enantiomer blocks the effect of ß1-receptors. The aims of this study were to develop a method for detecting and quantifying Clb and its enantiomeric distribution in several bovine tissues. The UHPLC-MS/MS method developed to quantify the target compound at trace levels in these tissues combines high sensitivity with good selectivity and short chromatographic run time. The tissue samples tested were found to contain racemic Clb in concentrations of 5-447 pg g-1 . The enantiomeric analysis of Clb showed that R-(-)-Clb is present at higher concentrations in some tissues, whereas S-(+)-Clb was detected in a ratio of 55/45 in the liver and heart tissues.

Chemical staining enhanced Enzyme-linked immunosorbent assay for sensitive determination of Clenbuterol in food.

Exploring a novel strategy for strengthening the catalytic activity of enzyme facilitates the development of a sensitive enzyme-linked immunosorbent assay (ELISA). Herein, a chemical staining (CS) strategy was firstly discovered to possess the ability to directly improve the catalytic activity of horseradish peroxidase. Based on this discovery, coomassie brilliant blue was introduced into ELISA to establish a CS enhanced ELISA (CS-ELISA) to detect clenbuterol (CL) by simply staining monoclonal antibodies. Satisfactorily, the most important analytical parameters of CS-ELISA, including sensitivity (0.074 ng mL-1) and linear range (0.2-2 ng mL-1) were all improving 2-folds compared with conventional ELISA. Moreover, the CS-ELISA shows good applicability in the detection of CL in pork tenderloin samples. The proposed CS-ELISA shows various advantages, such as cost-effective, easily accessible, enhanced catalytic activity of enzyme, higher sensitivity, and broader linear range, providing a new insight into enhanced ELISA for food safety.

Rapid Detection of Clenbuterol Residues in Pork Using Enhanced Raman Spectroscopy.

Clenbuterol (CB) is a synthetic ß-receptor agonist which can be used to improve carcass leanness in swine, but its residues in pork also pose health risks. In this report, surface-enhanced Raman scattering (SERS) technology was used to achieve rapid detection and identification of clenbuterol hydrochloride (CB) residues. First, the effects of several different organic solvents on the extraction efficiency were compared, and it was found that clenbuterol in pork had a better enhancement effect using ethyl acetate as an extraction agent. Then, SERS signals of clenbuterol in different solvents were compared, and it was found that clenbuterol had a better enhancement effect in an aqueous solution. Therefore, water was chosen as the solvent for clenbuterol detection. Next, enhancement effect was compared using different concentration of sodium chloride solution as the aggregating compound. Finally, pork samples with different clenbuterol content (1, 3, 5, 7, 9, and 10 µg/g) were prepared for quantitative analysis. The SERS spectra of samples were collected with 0.5 mol/L of NaCl solution as aggregating compound and gold colloid as an enhanced substrate. Multiple scattering correction (MSC) and automatic Whittaker filter (AWF) were used for preprocessing, and the fluorescence background contained in the original Raman spectra was removed. A unary linear regression model was established between SERS intensity at 1472 cm-1 and clenbuterol content in pork samples. The model had a better linear relationship with a correlation coefficient R2 of 0.99 and a root mean square error of 0.263 µg/g. This method can be used for rapid screening of pork containing clenbuterol in the market.

Presence of ß2 -agonist growth promoters in human urine samples: GC-MS/MS evaluation of the excretion profiles of ractopamine administered in microdoses.

ß2 -adrenergic agonists having the potential to be misused to enhance performance for their thermogenic and anabolic properties are prohibited in sports. Clenbuterol, ractopamine and zilpaterol are utilised legally or illegally as growth promoters of animals raised for their meat. No withdrawal times are imposed for ractopamine prior to slaughter; residues are detected in meat of treated animals, which constitutes a risk of inadvertent consumption. Insufficient information is available on the fate of ractopamine in humans to implement efficient detection in athletes' urine samples. We have developed a confirmation procedure for total ractopamine in urine following the enzymatic hydrolysis of glucuronides and sulphates and the conversion to tri-TMS derivative (limit of identification at 0.15 ng/ml). The sulphates were found to form between 85% to 97% of ractopamine excreted in athletes' urine samples analysed routinely or in volunteers following the administration of a micro-dose of 2.5 µg. Peak levels were reached at 2 to 6 h and decreased rapidly below 1 ng/ml 10 h after dosing. With one exception, the highest level estimated in athletes' samples was 1.2 ng/ml. Zilpaterol was confirmed in a few urine samples collected in the USA and Mexico (highest level 2 ng/ml), while hundreds of athletes' samples were reported to contain clenbuterol by our laboratory over the past 7 years. Most of these cases originated from Mexico (n = 102) and Guatemala (n = 119), often clustered in events during which multiple samples were collected, and for the vast majority, in levels lower than 0.2 ng/ml.

Magnetic Nanotag-Based Colorimetric/SERS Dual-Readout Immunochromatography for Ultrasensitive Detection of Clenbuterol Hydrochloride and Ractopamine in Food Samples.

Direct and sensitive detection of multiple illegal additives in complex food samples is still a challenge in on-site detection. In this study, an ultrasensitive immunochromatographic assay (ICA) using magnetic Fe3O4@Au nanotags as a capture/detection difunctional tool was developed for the direct detection of ß2-adrenoceptor agonists in real samples. The Fe3O4@Au tag is composed of a large magnetic core (~160 nm), a rough Au nanoshell, dense surface-modified Raman molecules, and antibodies, which cannot only effectively enrich targets from complex solutions to reduce the matrix effects of food samples and improve detection sensitivity, but also provide strong colorimetric/surface-enhanced Raman scattering (SERS) dual signals for ICA testing. The dual readout signals of the proposed ICA can meet the detection requirements in different environments. Specifically, the colorimetric signal allows for rapid visual detection of the analyte, and the SERS signal is used for the sensitive and quantitative detection modes. The proposed dual-signal ICA can achieve the simultaneous determination of two illegal additives, namely, clenbuterol hydrochloride and ractopamine. The detection limits for the two targets via colorimetric and SERS signals were down to ng mL-1 and pg mL-1 levels, respectively. Moreover, the proposed assay has demonstrated high accuracy and stability in real food samples.

Post-mortem investigation into a death involving doping agents: The case of a body builder.

INTRODUCTION: A young male was found dead on the bed of a hotel room. He was expected to take part in a bodybuilding competition the day after. During the site inspection, drugs of different types were found. The next day, an autopsy was performed. The evidence of cardiomegaly with organ congestion involving lung, liver, kidneys, adrenal glands, spleen and brain was confirmed by both the autoptic and the histopathological exam. However, the cause of death needed to be investigated. METHODS: A thorough toxicological investigation was undertaken by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS) and liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on samples of urine, blood and hair. RESULTS AND DISCUSSION: Clenbuterol, a long-acting selective beta2 agonist, was found in both blood (1 ng/ml) and urine (1 ng/ml), and evidence of its use was provided by the analysis of the 3-cm hair (25 pg/mg). The main metabolite of drostanolone (2 alpha-methyl-androsterone), an anabolic steroid, was found in the urine (202 ng/ml), where an increased ratio of testosterone/epitestosterone (T/E = 11) emerged. Due to the results of the hair analysis, a long-term use of various anabolic steroids was supposed. The integrated analysis of the results and the absence of other possible causes (such as trauma or cardiac conduction anomalies) led to the identification of the abuse of doping substances as the underlying cause of death. CONCLUSION: Hair analysis has proven to be crucial in identifying drug misuse and the contributing cause of death.

Electromembrane extraction of clenbuterol from swine urine for monitoring illegal use in livestock.

The illegal use of clenbuterol seriously endangers food safety and human health. Accurate monitoring of the illegal use of clenbuterol in livestock can efficiently prevent the clenbuterol residue pork products from entering the consumer market. Thus, in this study, a simple, rapid, and sensitive method for the determination of clenbuterol in swine urine was developed using electromembrane extraction combined with liquid chromatography-tandem mass spectrometry. It should be noted that the electromembrane extraction method presented many advantages of simple operation, fast mass transfer rate, good sample clean-up capability, and less organic solvent consumption. The effect of important factors on the extraction efficiency of clenbuterol was investigated. Under the optimal conditions, good linearity was achieved for clenbuterol over the range of 1-1000 ng/ml (linear correlation [R2 ] = 0.9996). The recoveries of clenbuterol in swine urine at three spiked levels ranged from 83.7% to 110.0% with relative standard deviation values lower than 9.7% (n = 4). The limits of detection and quantification for clenbuterol were 0.07 and 0.25 ng/ml, respectively. These results suggested that the proposed method has great potential for the extraction and determination of trace analyte in a complex sample matrix for monitoring illegal use in livestock.

Simultaneous determination of eight ß-adrenergic agonists in human urine by an isotope dilution-online clean-up system coupled with liquid chromatography-tandem mass spectrometry.

ß-Adrenergic agonist compounds are medicines that open up the lung's medium and large airways. ß-Adrenergic agonist compounds have been illegally or legally used to increase lean muscle mass in meat animals, bodybuilding, weight-loss programs, and athletes. Developing a rapid analytical approach for determining ß-adrenergic agonist compounds in biological samples is crucial for individual exposure assessment. This study established an analytical method for simultaneously measuring eight ß-adrenergic agonist compounds in human urine, including clenbuterol, terbutaline, salbutamol, ractopamine, zilpaterol, cimaterol, tulobuterol, and fenoterol. Two hundred microliters of a urine sample were added to eight deuterium-labeled internal standard mixtures and glucuronidase/arylsulfatase for enzymatic hydrolysis, and were then analyzed using an online clean-up system coupled with a liquid chromatography-tandem mass spectrometry system (LC-MS/MS). The limit of quantification ranged from 0.03 to 0.12 ng/mL urine for the eight ß-adrenergic agonist compounds. The relative standard deviations (RSD) of the within-run and between-run precisions were less than 10%, and the relative accuracy errors were less than 17% in the three-level spiked artificial urine samples. Two hundred eighty human urine samples collected from the general population in Taiwan were assessed to demonstrate the capability and feasibility of this method. The detection frequencies were 33% for clenbuterol, 5% for ractopamine, and less than 5% for the others. We concluded that the isotope dilution-online clean-up system coupled with LC-MS/MS method is a valuable analytical method for investigating urinary ß-adrenergic agonist compounds in humans and is valuable for human biomonitoring studies.

Detection of clenbuterol residues in beef sausages and its enantiomeric analysis using UHPLC-MS/MS: A risk of unintentional doping in sport field.

Clenbuterol (Clb) can be present in Mexico often but not all over the world in animal tissues and organs, therefore, potentially is derived from animal sources as well. The aims of this study were to develop and validate a method for detecting traces of clenbuterol in beef sausages. A calibration curve showed linearity in the range of 20-500 pg ml-1 . The limit of detection (LOD) and lower limit of quantification (LLOQ) were 5 and 10 pg g-1 , respectively. The analyte recovery was from 95.70% to 100.40% with an intraday relative standard deviation (RSD%) of 0.99%-2.10% and an interday RSD% of 0.54%-2.34%, R2 = 0.9998. The methodology developed was applied successfully in 15 samples of beef sausage, and 73.3% of the samples tested contained racemic clenbuterol in concentrations between 30 and 471 pg g-1 . The UHPLC-MS/MS method developed combines high sensitivity with good selectivity and short chromatographic run time. Additionally, the enantiomeric analysis of clenbuterol performed in beef sausages showed a 59% for R-(-)-Clb and 41% for S-(+)-Clb. The presence of clenbuterol in beef sausages could represent a risk of unintentional doping in sport field, because the clenbuterol is a banned substance included in the World Anti-Doping Agency's (WADA) list of prohibited substances.

Effects of long-term treatment with dietary theobromine on rat skeletal muscles.

BACKGROUND: Plastic changes of skeletal muscles, such as hypertrophy and atrophy, are dependent on physiological activities and regulated by a variety of signaling pathways, including cyclic adenosine monophosphate (cAMP) pathway. The cAMP inducing agents, such as the ß2-adrenergic agonist clenbuterol, are known to induce muscle hypertrophy, and has been reported to induce slow-to-fast transitions in rat soleus muscle. Theobromine, one of the active components of cacao, functions as an inhibitor of phosphodiesterase and increases cAMP. This study hypothesized that theobromine, like clenbuterol, can induce muscle hypertrophy and influence contractile properties. METHODS AND RESULTS: Male Wistar rats were fed a normal diet or a diet containing 0.05% theobromine for 20 weeks. Using biochemical, anatomical, and physiological techniques, effects of dietary theobromine on skeletal muscles (soleus, extensor digitorum longus, plantaris, and gastrocnemius) were examined. There were no significant differences in body weight, serum levels of proteins and lipids, muscle weights, dry/wet ratio of muscle weights, mitochondrial oxidation enzyme activity of muscles, isometric contractile properties of muscles, and muscle fatigue between control and theobromine-fed rats. Quantitative analysis of mRNA, however, revealed upregulation of myosin heavy chain 2x and myogenic differentiation 1, as previously reported in clenbuterol-treated muscles. CONCLUSION: The long-term theobromine (0.05%) diet in rats had no effect in inducing muscle hypertrophy and in changing contractile properties, although it had some similar effects of clenbuterol on muscle gene expression.

A new potentiometric platform: Antibody cross-linked graphene oxide potentiometric immunosensor for clenbuterol determination.

An immunosensor is based on the signal measurement obtained upon the reaction of an antibody antigen complex. It plays a significant role in various fields such as environmental analysis, production monitoring, drug detection or screening, veterinary medicine, and animal food. In this study, an antibody crosslinked graphen oxide (GO)-based potentiometric sensor has been developed for fast, simple, and economical detection of clenbuterol. In this context, the photosensitive amino acid bound GO platform is synthesized and used for the preparation of electrode material. Then, polymeric structure is characterized by infrared spectroscopy, and the performance of immunonano platform prepared by potentiometric sensor is evaluated. The effect of pH, response time, selectivity, and sensitivity is investigated. Under the optimized conditions, a simple and rapid method for the determination of clenbuterol from milk sample is established by immuno-potentiometric sensor. The detection limit has found to be 0.87 × 10-9 mmol L-1 for this immuno-potentiometric sensor. This immuno-potentiometric sensor has optimum pH at 7.0, a wide linear response (1.0 × 10-2 to 1.0 × 10-9 mmol L-1 ), rapid response time (2 Min) and 36 weeks operational lifetime.

Developing a Simple Immunochromatography Assay for Clenbuterol with Sensitivity by One-Step Staining.

An innovative lateral flow competition immunoassay (LFCIA) for detecting clenbuterol (CL) was developed by employing the advantages of the coomassie brilliant blue (CBB) staining method. An antibody stained by CBB was used both as a recognition reagent and as a chromogenic probe, enabling the simple but sensitive LFCIA of CL. The CBB-based LFCIA exhibited sensitivity for CL with a detection limit of 2 ng mL-1. Furthermore, this strategy was preliminarily verified by screening for CL in milk, pork tenderloin, and swine liver with recoveries ranging from 81 to 102%. Compared with conventional LFCIAs, the use of CBB as a signal label not only avoided the complicated material synthesis and surface modification process but also simplified the cross-linking with antibodies, meanwhile reducing the steric hindrance and increasing the possibility of immune recognition reactions, which was propitious for the effective utilization of antibodies. Taking advantages of the simplicity, rapidity, and cost-effectiveness, the CBB-based LFCIA may have potential for on-demand monitoring of general harmful small molecules by changing the kind of the staining antibody.

Ratiometric Fluorescent Probe Based on Diazotization-Coupling Reaction for Determination of Clenbuterol.

In view of the potential harm caused by illegal feeding of clenbuterol (CLB) in the livestock industry, herein, a novel ratiometric fluorescent probe based on graphene quantum dots (GQDs)@[Ru(bpy)3]2+ was elaborately constructed for CLB detection. In this probe, GQDs acted as response signals, and their fluorescence was remarkably quenched by CLB through the diazotization-coupling reaction. As for [Ru(bpy)3]2+ as a reference signal, its fluorescence was hardly affected. The intensity ratio of two fluorophores showed good linearity with CLB concentration in the range of 0.05-40 µM, accompanied by visualization of fluorescence variation from yellow to red. The detection limit was as low as 0.029 µM. Particularly, the probe was successfully used to detect CLB in pork and beef samples with satisfactory recoveries. To our knowledge, this is the first report on a ratiometric fluorescent probe for the detection of CLB, which possesses broad application prospects in food safety risk monitoring.

Sensitive detection of clenbuterol by hybrid iridium/silicon nanowire-enhanced laser desorption/ionization mass spectrometry.

There is increasing demand for anti-doping drug monitoring in sports and food safety checks by developing sensitive and fast analytical methods. Here we report the development of hybrid Ir/SiNW as a new MALDI matrix for the detection of small molecules. This matrix is characterized by sufficient UV absorption, low-noise background, and high efficiency in ionization of small molecules. Sensitive detection of clenbuterol (LOD: 0.18 pmol) and a variety of other small molecules has been achieved using the Ir/SiNW matrix with a reproducible performance. Compared to the individual components separately, the matrix of hybrid Ir/SiNW synthesized via in situ growth can promote the MS signal intensity by up to 10 fold under identical experimental conditions. We provide a unique mechanism for the high performance of the hybrid Ir/SiNW matrix with the characteristic properties of hydrogen atom transfer and enhanced protonation at the interface of the hybrid nanostructures. Our approach of using a hybrid Ir/SiNW matrix enables detection of clenbuterol quantitatively in complicated biological samples and in vivo experiments, promising a useful tool for food security and anti-doping drug monitoring in sports.

Single-dose administration of clenbuterol is detectable in dried blood spots.

Clenbuterol is a ß2 -agonist prescribed for asthmatic patients in some countries. Based on its anabolic and lipolytic effects observed in studies on rodents and in livestock destined for food production, clenbuterol is abused by bodybuilders and athletes seeking leanness. Urinary clenbuterol analysis is part of routine doping analysis. However, the collection of urine samples is time-consuming and can be intimidating for athletes. Dried blood spot (DBS) appears attractive as an alternative matrix, but the detectability of clenbuterol in humans through DBS has not been investigated. This study evaluated if clenbuterol could be detected in DBS and urine collected from six healthy men after oral intake of 80 µg clenbuterol. The DBS and urine samples were collected at 0, 3, 8, 24, and 72 h post-ingestion, with additional urine collections on days 7 and 10. Using LC-MS/MS, it was shown that clenbuterol could be detected in all DBS samples for 24 h post-ingestion and with 50% sensitivity 3 days after ingestion. The DBS method was 100% specific. Evaluation of analyte stability showed that clenbuterol is stable in DBS for at least 365 days at room temperature when using desiccant and avoiding light exposure. In urine, clenbuterol was detectable for at least 7-10 days after ingestion. Urinary clenbuterol concentrations below 5 ng/mL were present in some subjects 24 h after administration. Collectively, these data indicate that DBS are suitable for routine doping control analysis of clenbuterol with a detection window of at least 3 days after oral administration of 80 µg.